A novel FAM83G variant from palmoplantar keratoderma patient disrupts WNT signalling via loss of FAM83G-CK1α interaction

Palmoplantar keratoderma (PPK) is a multi-faceted skin disorder characterized by the thickening of the epidermis and abrasions on the palms and soles of the feet. Among the genetic causes, biallelic pathogenic variants in the FAM83G gene have been associated with PPK in dogs and humans. Here, a novel homozygous variant (c.794G>C, p.Arg265Pro) in the FAM83G gene, identified by whole exome sequencing in a 60-year-old female patient with PPK, is reported. The patient exhibited alterations in the skin of both hands and feet, dystrophic nails, thin, curly and sparse hair, long upper eyelid eyelashes, and poor dental enamel. FAM83G activates WNT signalling through association with ser/thr protein kinase CK1α. When expressed in FAM83G−/− DLD1 colorectal cancer cells, the FAM83GR265P variant displayed poor stability, a loss of interaction with CK1α and attenuated WNT signalling response. These defects persisted in skin fibroblast cells derived from the patient. Our findings imply that the loss of FAM83G-CK1α interaction and subsequent attenuation of WNT signalling underlie the pathogenesis of PPK caused by the FAM83GR265P variant.


Introduction
Palmoplantar keratoderma (PPK) is a heterogeneous skin disorder [1].Patients commonly display thickening of the skin on the palms and soles of the feet, which can be accompanied by other symptoms.Severe PPK is also associated with Olmsted syndrome, where patients have digit deformities on account of extremely thickened skin, which can lead to autoamputation [2].Hereditary PPK can be identified at infancy or in young adults as diffuse, focal or striated patterns of hardened thick skin that is often reported to be painful [3].Variants in keratin genes have been identified in PPK patients, including KRT1, which encodes keratin-1 in the differentiating suprabasal epidermis at the outer layer of the skin [4], and KRT16, which encodes keratin-16 in proliferating keratinocytes [5][6][7].Several variants in the cadherin-type protein desmoglein 1 (DSG1), which is abundant in the upper layer of epidermis, have also been reported in PPK patients [8].FAM83G missense variants were reported in two siblings [9] and a breed of dogs [10] with PPK.The two siblings, carrying c.101C>A, p.(Ala34Glu), missense mutation leading to the expression of FAM83G A34E protein, presented with thickened and damaged skin in the palms and soles of their feet and exuberant hair [9].Similarly, a missense variant in FAM83G leading to the expression of FAM83G R52P protein in dogs led to a diagnosis of hereditary footpad hyperkeratosis [10], with the reported phenotypes similar to human patients with PPK [9].Interestingly, transgenic mice that had a substantial deletion of the FAM83G gene locus displayed spontaneous woolly hair phenotype reminiscent of PPK patients and dogs [11].The exact molecular causes of hereditary and sporadic PPK remain to be fully elucidated.
FAM83G belongs to the FAM83 family of poorly characterized proteins that anchor isoforms of the CK1 family of ser/thr kinases to specific subcellular compartments through the conserved DUF1669 domain [12,13].The CK1 family of kinases regulates a plethora of cellular processes, including circadian rhythms, WNT signalling and cell division cycle [14].It has been shown that FAM83F-CK1α and FAM83G-CK1α interaction is critical for the activation of canonical WNT signalling [15,16], and FAM83D-CK1α interaction at the mitotic spindle is essential to ensure proper spindle positioning and timely cell division [17].The two reported PPK mutations, FAM83G A34E and FAM83G R52P in humans and dogs, respectively, were both previously shown to abolish the interaction with CK1α and led to attenuation of WNT signalling in Xenopus embryos and human cell lines [18].Here, a novel FAM83G homozygous variant (c.794G>C) was identified from a 60-year-old female patient diagnosed with PPK which causes Arg265 to Pro substitution (p.Arg265Pro) on the FAM83G protein.Detailed medical indications of the patient are presented, including images of the affected areas.A thorough molecular and functional characterization of the FAM83G R265P variant was also performed in DLD1 colorectal cancer cells as well as skin fibroblasts derived from the patient.The findings show that the loss of CK1α binding and subsequent inhibition of WNT signalling appear to underlie the pathogenesis of PPK caused by the FAM83G R265P mutation in this patient.Not only does this widen our understanding of how FAM83G-CK1α complexes function in WNT signalling but reinforces the significance of dysregulated WNT signalling to the development of PPK.

DNA sequencing analyses
Given the patient's clinical presentation (PPK), she was referred from the dermatology unit of Vall d'Hebron Hospital to the Clinical Genetics Unit of Vall d'Hebron Hospital to consider a genetic study.Therefore, the inclusion criteria for the patient's whole-exome study were as follows: a patient with suspected genetic disease (PPK) and multiple known genes associated with the pathology.DNA was extracted from peripheral blood cells derived from the proband (affected patient), both parents and two unaffected sisters following written informed consent, which was approved by the Vall d'Hebron University Hospital ethics committee.Single exome sequencing was performed in the proband using the xGen Exome Panel v2.0 (IDT).Sequencing was performed on an Illumina Novaseq6000 with 100 bp paired-end reads.Reads were aligned to the GRCh37 reference genome using Burrows-Wheeler aligner (version 0.7.3); duplicate reads were marked using Picard MarkDuplicates (version 2.20.8) and aligned reads were then processed using GATK BaseRecalibrator (Genome Analysis Toolkit; version 3.8) to recalibrate base quality scores, according to GATK Best Practices recommendations.Variants were filtered with a minor allele frequency of <0.005 based on gnomAD (Genome Aggregation Database, version 2.0) and prioritized based on the expected model of inheritance (autosomal recessive or de novo following autosomal dominant inheritance) and described gene-disease associations, using HPO terms and literature review of candidate genes.All prioritized variants were validated in the proband and segregated on her two unaffected sisters by Sanger sequencing.

Plasmids
All plasmids used in this study are listed below (See table 1).

Generation of skin fibroblasts from patients
Skin-punch biopsy was obtained after written informed consent, which was approved by the Vall d'Hebron University Hospital ethics committee, from the PPK patient (FIB04) as well as from two sex-and age-matched individuals with no PPK diagnosis (FIB03 and FIB15).Primary skin fibroblast cultures were established from the PPK patient and controls.Fibroblasts were cultured in Eagle's minimal essential medium (MEM, Thermofisher Scientific) supplemented with 20% fetal bovine serum (FBS, Gibco), 1% HEPES buffer 1 M (Cytiva), 1% non-essential amino acids (Corning) and 1% penicillin/streptomycin (Gibco).Cultures were established in T25 flasks and then split in T75 and maintained at 37°C in a 5% CO 2 atmosphere until passage number 4. Cell dissociation for passaging was performed with trypsin-EDTA (Sigma) treatment when cultures reached >95% confluency.

Generation of stable cell lines by retroviral transduction
pBabeD puromycin vectors (6 µg) were co-transfected with VSV-G (2.8 µg) and gag/pol (3.2 µg) plasmids into a 10 cm diameter dish of ~70% confluent HEK293FT cells.Plasmids were mixed with 24 µL PEI (1 mg ml −1 ) in 1 ml Opti-MEM and incubated at room temperature for 20 min before the mixture was added dropwise to HEK293FT cells.After 24 h of plasmid transfection, fresh DMEM was added to the cells.After 48 h of transfection, viral media were collected using a syringe and filtered through a 0.45 µm filter into a falcon tube.Target cells (70% confluent) were transduced with retroviruses diluted in fresh medium (1 : 2) and polybrene (8 µg ml −1 ) for 24 h.The transduction medium was then replaced with fresh medium supplemented with puromycin (2 µg ml −1 ) for selection of transduced cells.

Immortalization of primary fibroblasts using SV-40 T-antigen expression
Retroviruses encoding SV-40 T-antigen were collected, passed through a 0.45 µm filter and added to target primary fibroblasts (p5) for 24 h as described above, except the selection was carried out with hygromycin (200 µg ml −1 ) until untransduced control cells died.Surviving cells were cultured in DMEM+20% FBS and taken forward as immortalized fibroblast cell stocks.

Preparation of L-and Wnt3A-conditioned medium
Mouse fibroblast L-cells and L-cells stably overexpressing Wnt3A were cultured for three days in 15 cm dishes and medium collected for control-conditioned medium (L-CM) and Wnt3A-conditioned medium (Wnt3A-CM), respectively.The conditioned media were filtered (0.45 µm) prior to stimulation of target cells.

Cell lysis
Cells were harvested by washing twice with ice-cold phosphate-buffered saline (PBS) before scraping cells into ice-cold NP-40 cell lysis buffer (50 mM Tris-HCl pH 7.5, 0.27 M sucrose, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate and 1% NP-40) supplemented with 1 × cOmplete protease inhibitor cocktail (Roche) and 1 mM DTT. Cell lysates were incubated on ice for 10 min and then clarified by centrifugation at 14 000 rpm for 20 min at 4°C.Protein concentration was measured by Bradford assay and samples were normalized using lysis buffer for further use.

Immunoblotting
Extracts (15-20 µg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membrane.Membranes were blocked in TBS-T (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.2% Tween-20) with 5% (w/v) non-fat milk (Marvel).Membranes were then incubated overnight with agitation at 4°C in 5% (w/v) BSA or non-fat milk/TBS-T with the appropriate primary antibodies at the indicated dilutions provided in Section 2.10.Membranes were then washed for 3 × 10 min with 1×TBS-T before incubation with a secondary antibody for 1 h at room temperature.After 3 ×10 min washes with 1×TBS-T, protein signal was detected using ECL (Merck) and ChemiDoc MP System (Bio-Rad).Quantification of protein signal was performed using Fiji 1.53q (ImageJ), and data were processed using Microsoft Excel before representative graphs were generated from numerical data using GraphPad Prism 9.

Immunoprecipitation
For anti-GFP immunoprecipitations (IPs), anti-GFP beads (MRC PPU Reagents and Services) were pre-equilibrated using NP-40 lysis buffer.After an aliquot of the lysate was stored as the Input sample, 10 µl pre-equilibrated beads (50% slurry) was mixed with the clarified lysate (1 mg protein) and incubated on rotation for 2 h at 4°C.For anti-CK1α IPs, anti-CK1α antibody (MRC PPU Reagents and Services, 1 µg mg −1 protein in lysate) were first incubated with lysate (250 µg protein) on rotation (16 h, 4°C) before 20 µl protein G sepharose beads were added (1 h, 4°C) to pull-down anti-CK1α antibody and co-precipitating proteins.
Beads were pelleted by centrifugation (1200 rpm, 5 min), post-IP supernatant was collected as flow-through, and beads washed 3 × 0.5 ml lysis buffer before adding 50 µl NuPAGE 1 × lithium dodecyl sulfate (LDS) sample buffer.Samples were eluted by boiling at 95°C for 10 min.Input, IP and flow-through samples were analysed by SDS-PAGE, transferred to PVDF membranes and western blotting.

Quantitative real-time PCR and primers
Cells were seeded onto 6-well plates at 70% confluency.Cells were washed once with 1 × PBS before incubation with either L-CM or Wnt3A-CM for 6 h.RNA was extracted from the cells using the RNeasy Mini Kit (74104, QIAGEN) and RNA concentration was quantified using NanoDrop (Thermo Fisher Scientific).cDNA synthesis was performed using 1 µg RNA with the iScript cDNA synthesis kit (Bio-rad).Each well of a 384-well quantitative real-time PCR (qRT-PCR) plate contained the following reaction mixture (10 µl): 2 µl cDNA (diluted 1 : 10), 50% (v/v) iQ-SYBR Green supermix (Bio-Rad), 2 µM forward primer and 2 µM reverse primer.Reactions were performed on a CFX384 machine (Bio-Rad).Primers: Axin2: forward-TA CACTCCTTATTGGGCGATCA, reverse-TTGGCTACTCGTAAAGTTTTGGT; GAPDH: forward-TGCACCACCAACTGCTTAGC , reverse-GGCATGGACTGTGGTCATGAG.Fold change was determined using the comparative Ct method (ΔΔCt) for Wnt target gene Axin2 expression normalized to the expression of housekeeping gene GAPDH.Microsoft Excel software was used for data processing and GraphPad Prism 9 was employed for statistical analysis and graph preparation.At least three biological replicates and three technical replicates for each condition were employed.

A novel homozygous variant identified in 60-year-old female patient diagnosed with PPK
The 60-year-old female PPK patient reported a history of uneventful pregnancy, birth and normal early development.She reported that she first exhibited symptoms, such as alterations in the skin of both hands and feet, her hair, and dystrophic nails at the age 3.She was diagnosed with PPK at this stage.In the intervening years, physical examination revealed recurring features including thin, curly and sparse hair, long upper eyelid eyelashes, poor dental enamel and PPK, indications that  1a).Her family history includes parental consanguinity and two unaffected siblings.Written informed consent, which was approved by the Vall d'Hebron University Hospital ethics committee, was obtained from the patient prior to obtaining and using clinical materials and photographs.Whole exome sequencing (WES) performed on peripheral blood DNA from the patient identified a homozygous FAM83G gene variant (c.794G>C, p.Arg265Pro; figure 1b).The homozygous variant identified using gnomAD version 4.0 was predicted to be uncertain according to REVEL (0.29) but pathogenic according to AlphaMissense (0.986).Sanger sequencing performed on two unaffected siblings were both heterozygous, with c.794G>C variant seen only on one of the alleles (figure 1b).These results suggested an autosomal recessive trait for the phenotypic manifestation.

FAM83G R265P variant displays reduced stability, inability to interact with CK1α and reduced WNT activity
A GFP-tag was introduced at the C-terminus of WT FAM83G and the FAM83G R265P mutant and both were expressed in FAM83G −/− DLD1 cells [19] using retroviral transductions (figure 2a).Analysis of protein expression by immunoblotting revealed slightly lower expression of FAM83G R265P -GFP in comparison to WT FAM83G-GFP (figure 2a).To assess whether this could be due to the reduced stability of the FAM83G R265P mutant, cells were subjected to cycloheximide (CHX) treatment to inhibit new protein synthesis and protein abundance was monitored at specific time points up to 24 h by immunoblotting.
Compared with WT FAM83G-GFP, which appeared stable over 24 h following CHX treatment, FAM83G R265P -GFP levels were dramatically reduced to about 50% by 6 h and 70% by 24 h following CHX treatment (figure 2b,c), suggesting reduced stability of the FAM83G R265P -GFP.As expected, c-myc levels reached almost undetectable levels within 1 h of CHX treatment and remained undetectable thereafter (figure 2b).FAM83G exists in a stable complex with CK1α in cells [12,16].Therefore, whether the pathogenic FAM83G R265P mutant could interact with CK1α was assessed.While anti-GFP immunoprecipitates (IPs) of WT FAM83G-GFP from cell extracts robustly co-precipitated endogenous CK1α, the FAM83G R265P -GFP IPs did not (figure 3a), suggesting the inability of the FAM83G R265P mutant to bind to CK1α.
FAM83G reference FAM83G is reported to activate canonical WNT signalling via its interaction with CK1α [16].Consistent with this, the levels of Axin2 transcripts observed in WT DLD1 cells, which display enhanced WNT activity owing to an inherent APC mutation [20], were attenuated in FAM83G −/− DLD1 cells (figure 3b).Restoration of WT FAM83G-GFP in FAM83G −/− DLD1 cells partially restored Axin2 mRNA expression but the FAM83G R265P -GFP mutant did not (figure 3b), indicating that the mutant failed to induce WNT activity.

FAM83G R265P protein instability and its inability to interact with CK1α persists in patient skin fibroblasts
Fibroblasts from skin biopsies of the PPK patient harbouring the homozygous FAM83G c.794G>C (protein FAM83G R265P ) variant (FIB04) were collected along with two age-and sex-matched controls (FIB03 and FIB15).Written informed consent, which was approved by the Vall d'Hebron University Hospital ethics committee, was obtained from the patients for the generation and use of skin fibroblasts prior to skin biopsies.For biochemical assays, all skin fibroblast types were first immortalized by introducing SV-40 T-antigen through retroviral transductions (figure 4a).In both FIB03 and FIB15 control fibroblast cells, FAM83G protein levels were comparable to those observed in WT DLD1 cells, but in FIB04 skin fibroblasts from the PPK patient FAM83G protein levels were substantially lower (figure 4b; electronic supplementary figure S1), indicating that the R265P mutation affects FAM83G protein stability as observed above.All fibroblast cells displayed lower levels of CK1α compared with WT DLD1 cells, however, FIB04 cells displayed even lower CK1α levels than FIB03 and FIB15 controls (figure 4b; electronic supplementary material, figure S1).FAM83F protein, which also activates WNT signalling through an interaction with CK1α [15], was not detected in any of the skin fibroblast cells (figure 4b).Next, FAM83G-CK1α interaction at the endogenous level in skin fibroblasts was assessed.FAM83G was detected in anti-CK1α IPs from control FIB03 and FIB15 fibroblast cell extracts but not from PPK FIB04 extracts (figure 4c).No FAM83G was detected in control anti-IgG IPs from any cell line and, as expected, only anti-CK1α IPs enriched CK1α from all three cell lines (figure 4c).These data confirm that the pathogenic FAM83G R265P variant renders it incapable of interacting with CK1α at the endogenous level.

Reduced WNT signalling response in skin fibroblast cells from the PPK patient
Next, the effect on WNT signalling activity in PPK fibroblasts by FAM83G R265P expression was interrogated.U2OS osteosarcoma, FIB03 and FIB04 cells were incubated in the presence of control or Wnt3A conditioned medium for 6 h, and the expression of WNT target gene Axin2 was measured by qRT-PCR.In both U2OS and FIB03 control cells, Wnt3A treatment induced a significant increase in Axin2 transcript levels (figure 5a).In contrast, in PPK patient FIB04 skin fibroblasts, Wnt3A did not induce any increase in Axin2 transcript levels compared with controls (figure 5a), suggesting that either the cells do not respond to Wnt3A stimulation or the transcription of WNT genes is impaired via disruption of WNT signalling.In all cells, including PPK patient-derived FIB04 cells, increased levels of active β-catenin [21] and LRP6 S1490 phosphorylation [22] were evident upon Wnt3A treatment compared with controls (figure 5b; electronic supplementary material, figure S2), suggesting that WNT signalling activation by Wnt3A was intact.As a secondary method to investigate the WNT signalling response, the cells were incubated with an inhibitor of GSK3β.In the absence of a WNT ligand, β-catenin is continually degraded through the sequential phosphorylation of β-catenin by CK1α and GSK3β [23,24].Therefore, inhibition of GSK3β activates WNT signalling by stabilizing β-catenin.In WT U2OS and control FIB03 skin fibroblasts, a significant increase in Axin2 transcript expression compared with control was observed after treatment with GSK3β inhibitor (figure 5c).In contrast, in FIB04 fibroblasts, the GSK3β inhibitor elicited only a slight increase in Axin2 transcript levels (figure 5c), further suggesting defective WNT signalling activity.Finally, whether restoring WT FAM83G in PPK patient-derived FIB04 fibroblasts could rescue WNT signalling was investigated.Indeed, compared with untransduced FIB04 skin fibroblasts, in FIB04 cells stably expressing WT FAM83G, Wnt3A induced a substantial increase in Axin2 mRNA levels (figure 5d), indicating that the presence of the FAM83G R265P mutation in FIB04 cells causes defective WNT signalling responses.Furthermore, FIB04 cells stably expressing FAM83G F296A , a well-established CK1α-binding deficient mutant [12], did not significantly rescue Axin2 levels to the same extent as the WT protein, demonstrating that restoration of WNT signalling activity was dependent on FAM83G-CK1α interaction.

Discussion
Here, a novel homozygous variant c.794G>C in the FAM83G gene of a PPK patient is described which encodes the expression of FAM83G R265P mutant protein.The patient displayed skin and hair phenotypes that were similar to those previously reported in two siblings harbouring the homozygous FAM83G A34E variant [9] and in dogs harbouring the homozygous FAM83G R52P variant [10].Further characterization of the FAM83G R265P mutant protein revealed that it displayed reduced stability, and an inability to interact with CK1α or activate the canonical WNT signalling pathway, similar to what was observed previously with FAM83G A34E and FAM83G R52P mutant proteins that also cause PPK in humans and dogs, respectively [18].Our findings strongly imply that the loss of CK1α binding and the subsequent attenuation of WNT signalling underlie the pathogenesis of PPK caused by FAM83G variants.Loss of FAM83G-CK1α interaction and defective WNT signalling at the endogenous level in skin fibroblasts derived from the PPK patient have been shown.The siblings of the patient, who displayed no symptoms of PPK, were heterozygous for the FAM83G c.794G>C variant, suggesting autosomal recessive transmission for disease pathology.Interestingly, restoration of the WT FAM83G protein in skin fibroblast cells derived from the PPK patient, which was intended to mimic a heterozygous trait displayed by unaffected siblings of the patient, was able to rescue defective WNT signalling evident in the patient cells.The precise molecular mechanism by which FAM83G-CK1α complexes regulate the WNT pathway, and maintain homeostasis of the cells, that eventually lead to the defects prevalent in PPK patients remain unresolved.Nonetheless, restoring the defective FAM83G-CK1α interaction and WNT signalling in affected areas of PPK patients could be a way to intervene against PPK pathology.It would also be interesting to investigate whether correction of the c.794G>C variant back to WT in skin fibroblast cells derived from the PPK patient by CRISPR/Cas9 genome editing could restore FAM83G stability, FAM83G-CK1α interaction and defective WNT signalling in these cells.
It is possible that the prognosis of PPK stems from dysregulated signalling between the epidermal, dermal and hypodermal layers of the skin leading to mistimed cell differentiation and proliferation [25].Tight spatiotemporal control along the regulatory axis of several pathways including WNT, BMP and Shh signalling is important to maintain the healthy regeneration and multi-layered structure of the skin [26].It is conceivable that FAM83G when mutated may lead to dysregulated WNT signalling within each layer of the skin, triggering improper skin development and an increased number of terminally differentiated cells on the outer epidermis which becomes thickened skin in PPK patients.Analysis of other WNT target genes in addition to Axin2, such as E-cadherin and fibronectin [27,28], in patient fibroblasts as well as profiling their physiological characteristics, may strengthen findings presented here and support a role for FAM83G R265P in defective skin development and pathogenicity of PPK.

Figure 1 .
Figure 1.A novel homozygous FAM83G variant identified in a patient with PPK.(a) Clinical photographs taken from the patient show dystrophic nails (left panel), hyperkeratosis on the foot (middle panel), and thin, curly and sparse hair (right panel-patient's face is blurred to obscure identifying features).(b) WES results from the patient displaying the genetic variant c.794G>C p.(Arg265Pro) within the FAM83G gene against a reference sequence and Sanger sequencing results in the unaffected siblings showing the heterozygous variant.

Figure 2 .
Figure 2. FAM83G R265P -GFP stably expressed in FAM83G −/− DLD1 cells has lower stability.(a) WT DLD1 cells or FAM83G −/− DLD1 cells or those transduced with retroviruses to stably express either FAM83G-GFP or FAM83G R265P -GFP were lysed and extracts (20 µg protein) resolved by SDS-PAGE before transferring onto PVDF membranes.The abundance of FAM83G-GFP and FAM83G R265P -GFP was confirmed using immunoblotting with anti-GFP and anti-FAM83G antibodies.β-actin was used as a loading control.(b) FAM83G −/− DLD1 cells or those expressing FAM83G-GFP and FAM83G R265P -GFP were treated with cycloheximide (100 µg ml −1 ) and lysed at the indicated time points after treatment.Extracts (20 µg protein) were resolved by SDS-PAGE before transferring onto PVDF membranes, which were subjected to immunoblotting with the indicated antibodies.(c) Western blot quantification of signals from (b) where FAM83G signal was quantified using Fiji 1.53q (ImageJ), normalized to GAPDH signal, and presented relative to 0 h CHX treatment control (n = 3, error bars represent mean ± s.d.).

FAM83GFigure 3 .
Figure 3. FAM83G R265P -GFP does not interact with CK1α and fails to activate the canonical WNT signalling pathway.(a) Extracts from FAM83G −/− DLD1 cells stably expressing WT FAM83G-GFP and FAM83G R265P -GFP were subjected to anti-GFP IPs.Input extracts (20 µg protein), IPs and post-IP flow-through extracts (20 µg protein) were resolved by SDS-PAGE, transferred to PVDF membranes, and analysed by immunoblotting using the indicated antibodies.(b) WT DLD1 cells or cells from (a) were lysed, followed by RNA isolation and cDNA synthesis.qRT-PCR was performed using the cDNA to measure Axin2 and GAPDH transcript levels.Axin2 mRNA levels were normalized to GAPDH expression for all samples.Individual data points are shown on a scatter graph with an overlay of the mean ± s.d.Statistical analysis was performed using an unpaired Student's t-test with Welch's correction, *p < 0.05 (n = 6).

7 Figure 4 .
Figure 4. Reduced abundance of FAM83G and CK1α levels and loss of FAM83G-CK1α interaction in skin fibroblasts derived from PPK patient.(a) Schematic outline of the protocol used for immortalization of human skin fibroblast cells using retroviral transduction and SV−40 T-antigen overexpression.Retroviruses encoding SV−40 T-antigen were produced in HEK293FT cells and employed to transduce cultured skin fibroblast cells at passage 5. Transduced cells were selected in hygromycin and surviving immortalized fibroblast cells were taken forward.(b) WT DLD1 cells, control skin fibroblasts derived from Marfan syndrome patients (FIB15, FIB03) and PPK patient skin fibroblasts (FIB04) were lysed and extracts (20 µg protein) resolved by SDS-PAGE and transferred to PVDF membranes, which were subjected to immunoblotting using the indicated antibodies.(c) Clarified cell extracts (1 mg total protein) for each indicated skin fibroblast cell line were incubated with either CK1α antibody or IgG control (1 µg mg −1 extract protein) for 16 h on a rotating platform.Protein G sepharose beads were added to isolate co-precipitating proteins.Input extracts and IPs were subjected to SDS-PAGE and transferred to PVDF membranes, which were analysed by immunoblotting using the indicated antibodies.

Figure 5 .
Figure 5. Reduced WNT signalling activity in skin fibroblasts derived from the PPK patient.(a) WT U2OS, control fibroblasts (FIB03) and PPK patient skin fibroblasts (FIB04) were incubated with L-CM or Wnt3A-CM for 6 h before RNA extraction, cDNA synthesis and analysis of Axin2 and GAPDH transcripts by qRT-PCR.Axin2 mRNA expression was normalized to GAPDH and the mean of three biological replicates was plotted as a bar graph ± s.d.(b) WT U2OS, control fibroblasts (FIB15, FIB03) and patient skin fibroblasts (FIB04) were incubated with L-CM and Wnt3A-CM for 6 h before lysis.Extracts (20 µg protein) were resolved by SDS-PAGE and transferred to PVDF membranes, which were analysed by immunoblotting using the indicated antibodies.(c) As in (a), except the cells were pre-treated with DMSO or the GSK3 inhibitor CHIR99021 (GSK3i) at 5 µM for 6 h before RNA extraction, cDNA synthesis and qRT-PCR.(d) WT U2OS, control fibroblasts (FIB03), patient skin fibroblasts (FIB04) and two PPK patient cell lines retrovirally transduced to overexpress either WT FAM83G or CK1-binding deficient mutant FAM83G F296A were incubated with L-CM or Wnt3A-CM for 6 h and processed as in (a) for qRT-PCR to measure normalized Axin2 mRNA expression.Statistical analysis was performed using two-way ANOVA with multiple comparisons, **p < 0.01, ns = no statistical significance (n = 3, error bars represent ± s.d.).

Table 1 .
All plasmids used in this study.
Details of all antibodies used in this study are listed below shown below (See table2).

Table 2 .
Details of all antibodies used in this study.
persist presently (figure